Immunopathogenetic features characterize the autoimmune inflammatory myopathies - polymyositis, dermatomyositis, and related diseases: lymphocytic destruction of muscle cells, and humoral autoimmunity distinguished by a striking set of disease-specific autoantibodies. Although the muscle cell destruction is mediated by lymphocytes, the autoantibodies, particularly those directed against the family of functionally related but structurally diverse aminoacyl-tRNA synthetases, seem to offer a useful window on the disease and have been the focus of much of this group's research for a number of years. Work on this project in the past year was unexpectedly held up when the person who was to replace Dr. Kanneboyina Nagaraju, who moved to Johns Hopkins, was prevented by personal circumstances from starting until the end of the fiscal year. Nevertheless, animal experiments begun by Dr. Nagaraju continued with his supervision of the work of a post-BAC fellow. Dr. Nagaraju had shown that the controllable, muscle specific up-regulation of MHC I in skeletal muscle of mice led to a clinical and histological myopathy with many, though not all of the features of human inflammatory myopathy. The major aim of the experiments done in the past year was to determine the extent to which the myopathic lesion found in the mice when MHC Class I is controllably up-regulated in muscle requires the presence of an active immune response. In the published experiments, the primary inflammatory infiltrate was macrophagic-monocytic; very few lymphocytes were seen. Autoantibodies, including anti-Jo-1, which is limited to myositis patients, were found, though there is little reason to think that they play a pathogenetic role in either the human or the murine myopathy. In the experiments, the myopathic strain - itself the product of a pair of transgenes, was bred to RAG knockout mice. RAG-/- mice bearing both transgenes were difficult to obtain, but the results in the small number of offspring so far studied suggests that an active immune response is not required. Analogous experiments on the beta-2 microglobulin-/- are not complete. Thus, the pathogenesis of the myopathy differs from the presumed pathogenesis in the human disease and may more resemble a so-called surplus protein myopathy. The finding of anti-Jo-1 autoantibodies in the model, therefore, has major implications for the origin of this immune response, supporting the possibility that some intrinsic property of the autoantigen along with the circumstances of its exposure to the immune system - in muscle undergoing damage, with an altered display of MHC on the surface of muscle cells - is necessary to stimulate autoantibody production. With this in mind, we have actively pursued the possibility that had been suggested by experiments showing that an aminoacyl-tRNA synthetases (tyrosyl-tRNA synthetases) might also have chemoattractant properties. In collaboration with Dr. Zack Howard and the Oppenheim lab in the NCI at Frederick, we have been systematically exploring the chemoattractant properties of several classes of molecules: aminoacyl-tRNA synthetases (some of which are autoantigens and others of which are not); other autoantigens and the granzyme B proteolytic fragments of some of those autoantigens; molecules known to have coiled-coil structure (some of which are autoantigens and some of which are not); and various control molecules. The reason for studying coiled-coil containing proteins is that Jan Dohlman had shown that this structural feature is found far more commonly in autoantigens than in other proteins. In all cases, we are using highly purified recombinant human proteins we have ourselves made or we have obtained from colleagues at NIH (Peter Steinert and Harris Bernstein) or elsewhere (Livia Casciola-Rosen and Antony Rosen of Johns Hopkins; David Yang of Georgetown; and Michael Hartlein of EMBL Grenoble). So far, we have identified chemokine activity of two aminoacyl-tRNA synthetases, both of which are target autoantigens in myositis. For one of them, it is clear from experiments with transfected cell lines that the molecule stimulates cells through the CCR5 receptor, a known receptor on dendritic cells. It is noteworthy that not only are several CCR5 chemokines found in muscle in myositis (shown in my lab by Dr. Elizabeth Adams and previously published), but CCR5 can be found on dendritic cells in myositic muscle (found by Dr. Nagaraju, but not yet published). For the other, a likely candidate receptor is currently being verified. These experiments will be paralleled by experiments just underway in my own lab to determine the direct effect of these molecules on dendritic cells. There is also an active collaboration between my lab and Rosen group at Hopkins, including Dr. Nagaraju, examining the autoantibodies raised to cytotoxic granule treated muscle cells (presumed granzyme B derived cleavage products). These experiments, which were begun while Dr. Nagaraju was in my lab and have been underway for several years, are nearing completion. These experiments all can be seen to support the view that the microenvironment in particular tissues is a major determinant of the autoantibody repertoire.